Human Biochemistry Volume 2 - Murray R. 1993

Special Topics
Blood Plasma and the Coagulation Process
Blood Coagulation

The cessation of bleeding following traumatic injury to Blood Vessels is known as hemostasis. There are four distinct phases of hemostasis: The first phase is the constriction of the injured vessel, which reduces blood flow to the area distal to the site of injury. The second phase involves The formation of a loose platelet plug, or white thrombus, at the injury site. Collagen exposed at the site acts as a binding center for platelets; upon binding, their internal Structure breaks down, releasing thromboxane and ADP. These substances, in turn, recruit additional platelets, thereby forming a loose temporary plug. The duration of this hemostatic phase is determined by bleeding time. The third phase is the formation of a red thrombus (blood clot). The fourth phase involves the partial or complete dissolution of the clot.

Three types of thrombi, or blood clots, are distinguished. The white thrombus is composed of platelets and fibrin, containing relatively few erythrocytes. It typically forms at sites of injury or on pathologically altered vessel walls under conditions of high blood flow velocity (in Arteries). The second type consists of disseminated fibrin deposits within very small vessels (capillaries).

The third type of thrombus—the red thrombus—consists of erythrocytes and fibrin. The Morphology of a red thrombus closely resembles that of blood clots formed in vitro. Red thrombi form in vivo in regions of sluggish BLOOD FLOW IN the absence of pathological changes to the vessel wall, either at the site of injury or upon an altered vessel wall

just downstream from the initiating platelet plug. The initiation of clot formation in response to tissue damage proceeds via the Extrinsic pathway of coagulation. Conversely, the initiation of red thrombus formation in areas of sluggish blood flow or on abnormal vascular surfaces without prior tissue injury occurs via the intrinsic pathway. Both the extrinsic and intrinsic pathways converge on a common final pathway. This stage involves The conversion of prothrombin to Thrombin and the thrombin-catalyzed transformation of fibrinogen into thrombin-derived fibrin.

Thrombin-Catalyzed Conversion of Fibrinogen to Fibrin

Fibrinogen¹ (Factor I, see Fig. 55.1 and Table 55.3) is a soluble plasma glycoprotein synthesized in the Liver; its molecular length is 46 nm and its molecular weight is 340,000. The molecule consists of six polypeptide chains (two Aα-chains, two Bβ-chains, and two γ-chains), giving it the subunit structure Aα₂Bβ₂γ₂. The Bβ- and γ-chains contain complex Oligosaccharides linked to Asn residues. All three genes encoding the Aα-, Bβ-, and γ-chains are linked, and their expression in humans is coordinately regulated. The ends of the fibrinogen molecule carry a strong negative charge, attributable to a high concentration of aspartate and glutamate residues in the A-region of the Aα-chain and the B-region of the Bβ-chain (Fig. 55.6). Additionally, the B-region of the Bβ-chain contains an unusual negatively charged Tyrosine O-sulfate residue. These negatively charged termini not only contribute to the high Water solubility of fibrinogen but also electrostatically repel the ends of adjacent fibrinogen molecules, thereby preventing spontaneous aggregation.

Class="center">Table 55.3. Blood coagulation factor numbering system. The numerical designations do not reflect the sequential order of their action

¹ All coagulation factors (with the exception of fibrinogen, prothrombin, their activation products, and Ca²⁺ ions) are designated by Roman numerals (Table 55.3).

Fig. 55.6. Schematic representation of fibrinogen, showing its (AαBβγ)₂ structure, charged termini, and the four Arg-Gly peptide bonds cleaved by thrombin (indicated by arrows).

Thrombin is a Serine protease with a Molecular Weight of 34,000, consisting of two polypeptide chains. It hydrolyzes four Arg-Gly peptide bonds in fibrinogen (Fig. 55.6). Of these four bonds, two connect regions A and a, while the other two connect regions B and β in the Aα- and Bβ-chains, respectively. The fragments A and B released from the fibrinogen molecule are negatively charged fibrinopeptides, leaving behind a fibrin monomer with the structure (αβγ)₂. These long, insoluble Fibrin monomers spontaneously assemble into regular staggered structures, yielding an insoluble polymeric fibrin clot. This network traps erythrocytes, platelets, and other blood elements, forming either a red thrombus or a white thrombus (platelet plug). At an early stage, the fibrin clot is a highly loose structure held together solely by non-covalent interactions among insoluble fibrin monomers.

Beyond converting fibrinogen to fibrin, thrombin Functions to activate Factor XIII into its active form (XIII_a). Factor XIII_a (a transglutaminase) cross-links fibrin monomers by forming specific isopeptide bonds between the γ-carboxamide group of glutamine and the ε-amino group of Lysine (Fig. 55.7). This stabilization of the fibrin clot promotes clot retraction, a phenomenon readily observed in vitro. The increased bleeding tendency seen in patients with hereditary Factor XIII deficiency is due to the inability to form a stable fibrin clot.

Sudden vascular thrombosis can have dangerous and even catastrophic consequences, which is why in vivo thrombin activity must be meticulously regulated. This control is achieved through two primary mechanisms. One mechanism relies on the thrombin antagonist antithrombin III (see below). The second mechanism involves the synthesis and Circulation of prothrombin, the catalytically inactive zymogen of thrombin. Prothrombin, or Factor II, is synthesized in The Liver and contains γ-carboxyglutamate (Gla) residues. It is a single-chain glycoprotein with a molecular weight of 72,000; Fig. 55.8 illustrates the primary and Introduction/11.html">Secondary structure of this molecule. The N-terminal region of prothrombin (labeled 1 in the diagram) contains up to 14 Gla residues. The dashed line denotes the disulfide bridge connecting the A and B regions of prothrombin. The black triangle marks the Location OF THE catalytically active serine residue within the protease Active Site.

Fig. 55.7. Cross-linking of fibrin molecules by activated Factor XIII.

Fig. 55.8. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF prothrombin. The N-terminus is on the left; region 1 contains all the Gla residues. Sites of Cleavage by Factor X_a and the names of the resulting cleavage products are indicated. THE POSITION OF the catalytically active serine residue is marked with ▲. The A and B chains of active thrombin (shaded) are held together by a disulfide bond.

Prothrombin activation takes place On the surface of platelets, a process requiring anionic platelet Phospholipids, Ca²⁺ ions, and Factors V_a and X_a.

Fig. 55.9. Diagram showing the binding of Factors V_a, X_a, Ca²⁺ ions, and prothrombin to the platelet Cell/33.html">Plasma Membrane.

Phospholipids located on the inner leaflet of the platelet plasma membrane become exposed As a result of collagen-induced platelet damage and degranulation. These phospholipids bind Ca²⁺ ions and prothrombin (the latter via its Gla-containing N-terminal region). Platelets also contain Factor V, which, upon activation to Factor V_a, binds to specific receptors on the platelet membrane (Fig. 55.9). Factor V_a acts as a receptor for Factor X_a, which in turn binds prothrombin at the F-1.2 domain (Fig. 55.8). Factor X_a, also a serine protease, cleaves the catalytically inactive prothrombin molecule at the sites indicated in Fig. 55.8, releasing the N-terminal portion. Subsequent cleavage of prothrombin by Factor X_a yields the thrombin A and B Polypeptides, which remain linked by a disulfide bridge.

Phospholipid binding via Ca²⁺ ions to the Gla residues of prothrombin accelerates its activation by 50- to 100-fold, likely by establishing a high local concentration of both prothrombin and Factor X_a (Fig. 55.9). Factor V_a further enhances prothrombin activation approximately 350-fold, likewise by increasing the local concentration of Factor X_a.

Factor V_a, generated from Factor V by the action of thrombin, is subsequently inactivated by that same thrombin, thereby serving as a feedback mechanism to limit the conversion of prothrombin to thrombin.

Prothrombin can be activated by staphylocoagulase via conformational changes.

Activation of Factor X

At the stage of factor X activation, the extrinsic and intrinsic pathways converge to form the common final pathway of blood coagulation (Fig. 55.10). Factor X is a serine protease zymogen with a molecular weight of 55,000 and contains Gla residues. As in prothrombin, the Gla residues of factor X ensure Ca²⁺-dependent binding to acidic phospholipids in platelet membranes. The conversion of factor X into its active form (X_a) requires the cleavage of an Arginine-isoleucine bond by another serine protease. Two serine proteases are known to cleave the Arg—Ile bond in the factor X molecule.

Extrinsic pathway of factor X_a formation

The Cleavage of the Arg—Ile bond, and consequently the conversion of factor X into factor X_a in the extrinsic pathway, is carried out jointly by factor VII_a and tissue factor. Factor VII_a functions exclusively in the extrinsic pathway, which is rapidly triggered following tissue injury. The precursor of factor VII_a—factor VII (another Gla-containing glycoprotein)—is synthesized in the liver and can be activated by thrombin or factor X_a. Although factor VII is a zymogen, it possesses a relatively high intrinsic activity. Tissue factor, which accelerates the action of factors VII or VII_a on factor X, is found in highest concentrations in the Placenta, Lungs, and Brain.

Fig. 55.10. Interrelationship of the intrinsic, extrinsic, and common final pathways in blood coagulation.

1 ml of plasma contains approximately 3 mg of fibrinogen and only 0.01 mg of factor X. This implies that the coagulation system must involve Amplification. Indeed, the conversion of factor X to X_a is an autocatalytic process that can be viewed as an amplification step. In the reaction group considered here, it is not easy to determine which comes first, the "chicken or the egg"; in this case, factor II_a (thrombin) or factor X_a (Fig. 55.10).

Intrinsic pathway of factor X_a formation

The intrinsic pathway of factor X_a formation begins with the in vivo interaction of prekallikrein, high-molecular-weight kininogen, and factors XII and XI on an activating surface, likely collagen (Fig. 55.11). Glass and kaolin serve as activating surfaces for the intrinsic pathway in in vitro experiments. The interaction of factor XII with the activating surface makes it more susceptible to proteolytic attack by kallikrein. Action of kallikrein yields factor XII_a, which in turn induces the conversion of prekallikrein to kallikrein, thus establishing a reciprocal activation mechanism. Factor XII_a releases bradykinin from high-molecular-weight kininogen and activates factor XI to XI_a. Through two consecutive reactions, factor XI_a activates factor IX (a Gla-containing zymogen). In the presence of Ca²⁺ ions and acidic phospholipids, factor IX_a slowly activates factor X; this activation occurs via the cleavage of the same Arg—Ile bond that is cleaved by factor VII_a in the extrinsic pathway. The rate of factor X activation by factor IX_a is increased 500-fold in the presence of factor VIII (or VIII_a). Factor VIII activation apparently requires a small amount of thrombin. Factor VIII is not a protease; it likely serves as a receptor for factor IX_a during the cleavage of the Arg—Ile bond in factor X. The intrinsic blood coagulation pathway is a slow process due to the involvement of numerous factors. Together, they form a cascade mechanism that generates factor X_a (Fig. 55.11).

Table 55.4 lists a series of inherited human disorders caused by deficiencies in various Components of the coagulation system. The most frequent is factor VIII deficiency, which determines hemophilia A (the corresponding Gene is localized to the tenth human chromosome). This disease has played a significant role in The history of European royal dynasties.

Fig. 55.11. Intrinsic pathway of factor X activation to X_a. HMWK — high-molecular-weight kininogen.

Table 55.4. Hemorrhagic disorders and associated defects

Patients with autosomal dominant von Willebrand disease exhibit impaired platelet adhesion In addition to factor VIII deficiency. In contrast, patients with hemophilia A lack only the procoagulant activity of factor VIII, while platelet adhesion remains unaffected. Platelet adhesion factor (von Willebrand factor) is synthesized by vascular endothelial Cells and megakaryocytes (platelet precursor cells); it is a large glycoprotein with a molecular weight exceeding 200,000. von Willebrand factor is found in plasma and platelets as a complex with factor VIII molecules. The platelet surface presumably contains a glycoprotein receptor that binds the von Willebrand factor–factor VIII complex. von Willebrand factor likely stabilizes the procoagulant activity of factor VIII. von Willebrand disease can result from an inherited defect in the oligosaccharide moiety of the glycoprotein von Willebrand factor. An abnormal oligosaccharide may impair platelet adhesion and destabilize factor VIII. In hemophilia A, the defect resides in factor VIII; its procoagulant activity is impaired, whereas platelet adhesion, determined by von Willebrand factor, remains unchanged. Factor VIII is a glycoprotein comprising 2,300 Amino Acids, and its molecule shows partial Homology with ceruloplasmin and factor V. This factor is synthesized in the liver, Spleen, and Kidneys.

Blood Coagulation Tests

To become familiar with various Methods used to assess the functioning of the BLOOD COAGULATION SYSTEM, readers are encouraged to consult relevant sections of textbooks on physiology or hematology.

Anticoagulants

Normal plasma exhibits several types of antithrombin activity. A minor contribution is made by a₁-antitrypsin. Specific a₂-globulin accounts for about 25% of the total antithrombin activity of plasma. It forms an irreversible complex with thrombin and other proteases, thereby preventing the binding of these Enzymes to their natural substrates. a₂-Globulin is regarded as an a₂-plasmin inhibitor because it also inactivates plasmin, a serine protease with fibrinolytic activity.

The greatest antithrombin activity is attributed to antithrombin III. Antithrombin III possesses negligible intrinsic activity and is strongly activated in the presence of highly negatively charged heparin. Heparin apparently binds to a specific cationic site on antithrombin III, inducing a conformational change in its molecule. As a result of this change, antithrombin III acquires The ability to bind all serine proteases, including Trypsin, Chymotrypsin, and plasmin. In the blood coagulation system, antithrombin III inhibits The activity of thrombin and factors IX_a, X_a, XI_a, and XII_a. Individuals with inherited antithrombin deficiency show a tendency toward thrombus formation. This leads to the Conclusion that antithrombin performs vital physiological functions and that normal human blood coagulation is a highly dynamic system.

Heparin is frequently used in clinical practice as an anticoagulant drug. The primary factor determining the anticoagulant activity of heparin is its activation of antithrombin III, which in turn inhibits the serine proteases discussed above. Small amounts of heparin are known to be present on vessel walls, thereby reducing the activation of the intrinsic pathway. The anticoagulant activity of heparin can be neutralized by strongly cationic polypeptides (e.g., protamine). Such polypeptides compete with the cationic sites of antithrombin III for binding to polyanionic heparin.

Coumarin drugs inhibit the vitamin K-dependent carboxylation of Glu residues, which leads to Gla formation in the N-terminal region of factors II, VII, IX, and X. All these factors are synthesized in the liver, and the formation of Gla residues is essential for their maturation and, consequently, for the normal functioning of the intrinsic, extrinsic, and common pathways of coagulation. Coumarin drugs apparently inhibit the reduction of vitamin K quinone derivatives to their active hydroquinone forms. Administration of vitamin K overcomes the coumarin-induced block and ensures the hepatic maturation of Gla-dependent coagulation factors. The reversal of coumarin action by vitamin K takes 12–24 hours, whereas the reversal of heparin's anticoagulant activity by protamine occurs almost immediately; this difference is due to The Nature of their antagonistic mechanisms.

Fibrinolysis

Compelling evidence indicates that the normal blood coagulation system exists in a dynamic equilibrium where fibrin clots are continuously formed and subsequently dissolved. Plasmin is a serine protease capable of hydrolyzing Fibrinogen and fibrin, factors V and VIII, Complement factors, and various polypeptide Hormones. Normally, plasmin is present in plasma in the form of an inactive proenzyme (plasminogen). Most body Tissues contain various types of plasminogen activators. Tissue plasminogen activator is a serine protease that is catalytically inactive in the absence of contact with fibrin. Upon contacting fibrin, plasminogen activator is able to cleave the plasminogen molecule to yield plasmin. When plasmin hydrolyzes fibrin, the plasminogen activator loses its activity, and proteolysis subsides. This ensures the efficient regulation of fibrinolysis. The THERAPEUTIC USE OF recombinant tissue plasminogen activator (tPA) appears highly promising. tPA helps restore coronary artery patency, thereby reducing myocardial damage occurring during acute coronary thrombosis. Another plasminogen activator, the proteolytic enzyme urokinase, is found in urine. Urokinase is also a serine protease; it can activate plasminogen by cleaving it at two sites.

Plasminogen normally co-precipitates with fibrin and is therefore incorporated into the fibrin clot. The plasmin generated upon activation cleaves fibrin molecules into soluble fragments, causing the clot to dissolve (disappear). Cross-linked fibrin clots are more resistant to plasmin Digestion.

The concentration of plasminogen activators increases in A number of conditions, including certain forms of Cancer and Shock. Antiplasmin activity, mediated by a₁-antitrypsin and a₂-plasmin inhibitor, may be reduced in liver cirrhosis. Certain bacterial products, such as streptokinase, are capable of activating plasminogen without cleaving its molecule and may be responsible for the diffuse hemorrhages occasionally observed in patients with disseminated bacterial infections.

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